CN112823012A - Organoid compositions for the production of hematopoietic stem cells and derivatives thereof - Google Patents

Abstract

The present disclosure relates to compositions derived from precursor cells, and methods of using such compositions to make Hematopoietic Stem Cells (HSCs) or derived immune cells. More specifically, methods are disclosed for obtaining hematopoietic stem cells from organoid tissue or a culture comprising organoids, wherein the organoid tissue or culture comprises liver or colon tissue derived from precursor cells (e.g., embryonic stem cells or induced pluripotent stem cells) by directed differentiation.

Description

Organoid compositions for the production of hematopoietic stem cells and derivatives thereof

Cross Reference to Related Applications

This application claims priority and benefit from U.S. provisional application serial No. 62/730,061 filed on 12.9.2018, the entire contents of which are incorporated herein by reference.

Background

Currently, bone marrow transplantation is the primary means of treatment in individuals requiring reconstitution of the hematopoietic system. Stem and progenitor cells in the donated bone marrow are able to proliferate and replace blood cells responsible for protective immunity, tissue repair, coagulation, and other functions of the blood. In a successful bone marrow transplant, blood, bone marrow, spleen, thymus, and other immune organ functions are able to repopulate with cells from the donor. Bone marrow has become increasingly successful in treating a variety of diseases, including certain types of anemia, such as aplastic anemia, fanconi anemia, immunodeficiency, cancer, such as lymphoma or leukemia, cancer, various solid tumors, and hematopoietic disorders. Bone marrow transplantation has also been used to treat hereditary storage disorders, thalassemia major, sickle cell disease, and osteoporosis.

Although Hematopoietic Stem Cells (HSCs) have the ability to differentiate into all types of blood cells and can be transplanted to treat blood disorders, it is difficult to obtain HSCs in large quantities due to the lack of donors. Furthermore, the use of bone marrow transplantation to provide immune cells to an individual in need is severely limited due to the small number of perfectly matched (genetically identical) donors.

Thus, there remains a need in the art for HSC compositions suitable for transplantation and methods of providing HSCs. Furthermore, the development of such compositions would be helpful for research purposes because of the insufficient number of HSCs currently available. The present disclosure seeks to address one or more of the above-mentioned needs in the art.

Disclosure of Invention

The present disclosure relates to compositions derived from precursor cells, and methods of using such compositions to make Hematopoietic Stem Cells (HSCs) or derived immune cells. More specifically, methods are disclosed for obtaining hematopoietic stem cells from organoid tissue or a culture comprising organoids, wherein the organoid tissue or culture comprises liver or colon tissue derived from precursor cells (e.g., embryonic stem cells or induced pluripotent stem cells) by directed differentiation.

Drawings

Those skilled in the art will appreciate that the drawings described below are for illustration purposes only. The drawings are not intended to limit the scope of the present invention in any way.

Fig. 1. Characteristic of Human Liver Organoid (HLO) gene expression at day 21 of culture a. albumin expression was moderately reduced, while alpha-fetoprotein (AFP) expression was increased, compared to previous methods of differentiating mature liver organoids (Ouchi et al, 2019). B. Endothelial markers CD34 and KDR (VEGFR2) were increased. C. Both Erythropoietin (EPO) and hemoglobin gamma (HBG) are increased.

Fig. 2. Differentiation of bone marrow line A from HLO cultures on days 8-20, HLO cultures were dissociated into single cell suspensions and plated on formazan containing cytokinesOn cellulose to promote bone marrow differentiation, colonies were analyzed 7-14 days later. B. Representative Giemsa staining showed that multiple cell types were generated. CFC colony quantification comparison of cells from HLO culture with Umbilical Cord Blood (UCB) CD34⁺Cells and undifferentiated ipscs (n.d. ═ undetectable).

Fig. 3. B cells were differentiated from HLO cultures. A. On days 8-20, cultures of HLO were dissociated into single cell suspensions and co-cultured with confluent MS-5 cells. B. After co-cultivation with MS-5, UCBCD34⁺Flow cytometry of cells and HLO. Cells were first gated on CD45, followed by CD19 and CD11B to isolate B cells and bone marrow cells, respectively.

Fig. 4. Hematopoietic endothelium co-develops in human colon organoid cultures. (A) Whole RUNX1 (red), endothelial mucin (green) and CDX2 (white) stained in the dorsal aorta e10.5 mouse embryo nuclei RUNX1 (n ═ 3). (B, C) optical sections from (A). (d) Complete RUNX1 (red), CD34 (green) and CDH1 (white) staining in 22-day-old HCO showed nuclear RUNX1 staining in CD34+ endothelial tubes (n ═ 3). (E, F) optical sections from (D). DA ═ the dorsal aorta. (G) Plots of TPM (per million transcripts) values from RNAseq data for 21 day old HIOs and HCOs (n-3 for each group). (H) Flow cytometric images of gated CD34+ cells stained with CD45 and CD 73. CD34+/CD45-/CD 73-cells were boxed and black. CD34+/CD45+/CD 73-cells were boxed and green.

Fig. 5. Erythro-myeloid and lymphoid progenitors are produced in HCO culture. (A) Photomicrographs of cytospin cells from HCO cultures. (B) In the method of^TMExamples of colony formation in the medium. HCO derived from (C) H1 human embryonic stem cells and (D) cells of IPSC 263-10 in Methocult^TMQuantification of colony formation. (E) Flow cytometric images of CD 45-gated cells stained with CD3 and CD4, with and without treatment with T cell differentiation inducing cytokines.

Fig. 6. HCO comprises co-developing macrophages. (A) Immunofluorescent staining of human colon biopsies counterstained with DAPI for CD163 (red) and CDH1 (green). (A') an illustration of the boxed area in (A). Full-size of (B) HIO and (C) HCO counterstained with DAPI for stained CD163 (Red) and CDH1 (Green). (D) Plots of TPM (transcripts per million) values for SPI1(pu.1) and (E) CD163, RNAseq data from 35 day old HIOs and HCOs (n-3 for each group). Counterstained with DAPI for hCD163 (red) and F4/80 (green) (F) mouse colon, (G) immunofluorescence staining of human colon biopsies and transplanted HCO.

Fig. 7. HCO has inflammatory macrophages capable of secreting pro-inflammatory cytokines. (A) Heatmaps of inflammation-associated genes were generated from RNAseq data for 35 day old HIO and HCO. (B) Immunofluorescence staining of 35 day old HCO counterstained with DAPI for CD163 (green), iNOS (red) and CDH1 (white). (B') an illustration of the boxed area in B, with the exception of DAPI. (C-D) Luminex array data for IL-6, IL-8, MIP1A (CCL3) and MIP1B (CCL 4). Each point represents the Luminex value from individual differentiation. Paired HIO and HCO samples (from the same differentiation) are indicated by lines.

Fig. 8. HCO macrophages are functional. (A) Time-course micrograph of in vivo imaging of LPS-treated HCO (B-E) Luminex array data for HCO or IL-6, IL-8, MIP1A (CCL3) and MIP1B (CCL4) of LPS-treated HCO. (F) -/+ pHRODO E.coli particles (green) and CD14 (red) by immunofluorescence staining with 35 day old HCO. (G) Quantification of phagocytic particles (green) (n ═ 3 well organoids per group).

Fig. 9. HCOMacs migrate into the lumen of the organ-like cavity in response to bacteria. Immunofluorescent staining with DAPI counterstained CDH1 (green) and MUC2 (red) 35 day old HCO 24 hours after injection of (a) PBS, (B) commensal escherichia coli and (C) EHEC. Immunofluorescent staining with DAPI counterstained CDH1 (green), HAM56 (red) and 35 day old HCO of e.coli 24 hours after injection of (D) PBS, (E) symbiont e.coli and (F) EHEC. (G) Quantification of MUC2 fluorescence intensity (n-3 for each set). (H) Quantification of fluorescence intensity of E.coli (n. multidot.3 for each group). (I) Quantification of HAM56 macrophage distribution (n-3 organoids per group).

Fig. 10. And (5) experimental work flow.

Fig. 11. BMP signaling designates the hematogenic endothelium.

Fig. 12. Endothelial cells and hematopoietic cells co-develop in HCO culture.

FIG. 13. Expression of hemoglobin in HCO culture derived red blood cells.

Fig. 14. Cellular fluorescence analysis of immune cells present in HCO.

Fig. 15. Wells fig. S6. Macrophages persist in HCO after transplantation into the mouse kidney capsule.

Fig. 16. Wells fig. S7. Gene ontology analysis revealed parallel cell differentiation, macrophage maturation and inflammation in HCO.

Fig. 17. Macrophages within HCO are capable of extending filopodia in response to e.

Fig. 18. Imaging of placenta, liver, lung and colon.

Detailed Description

Definition of

Unless otherwise indicated, the terms are to be understood in accordance with their ordinary usage by those of ordinary skill in the relevant art. In the event of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods and reference to "a dose" includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which error range will depend in part on how the value is measured or determined, such as the limitations of the measurement system. For example, "about" can mean within 1 or greater than 1 standard deviation, according to practice in the art. Alternatively, "about" may refer to a range of up to 20%, or up to 10%, or up to 5%, or up to 1% around a given value. Alternatively, particularly for biological systems or processes, the term may refer to a value within an order of magnitude, preferably within 5-fold, more preferably within 2-fold. Where particular values are described in the application and claims, unless otherwise stated, the term "about" should be assumed to be within an acceptable error range for the particular value.

As used herein, the term "totipotent stem cell" (also referred to as "universal stem cell") is a stem cell capable of differentiating into embryonic cells and extra-embryonic cell types. Such cells are capable of constructing complete, viable organisms. These cells are produced by fusion of egg and sperm cells. The cells resulting from the first few divisions of the fertilized egg are also totipotent.

As used herein, the term "Pluripotent Stem Cell (PSC)", also commonly referred to as PS cell, includes any cell capable of differentiating into almost all cells, i.e., cells derived from any of the three germ layers (germ epithelium) [ including endoderm (gastric lining, gastrointestinal tract, lung), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epidermal tissue and nervous system) ]. PSCs can be progeny of totipotent cells, derived from embryonic stem cells (including embryonic germ cells) or by inducing non-pluripotent cells (such as mature somatic cells), obtained by forced expression of certain genes.

As used herein, the term "Induced Pluripotent Stem Cell (iPSC), also commonly abbreviated as iPS cell, refers to a class of pluripotent stem cells that are artificially derived from non-pluripotent cells in general, such as adult somatic cells, by inducing" forced "expression of certain genes.

As used herein, the term "Embryonic Stem Cell (ESC)", also commonly abbreviated as ES cell, refers to a pluripotent cell derived from the inner cell mass of a blastocyst (i.e., early embryo). For the purposes of the present invention, the term "ESC" is sometimes used broadly to encompass embryonic germ cells.

As used herein, the term "precursor cell" encompasses any cell that can be used in the methods described herein by which one or more precursor cells acquire the ability to renew themselves or differentiate into one or more specific cell types. In some aspects, the precursor cells are pluripotent or have the ability to become pluripotent. In some aspects, the precursor cells are treated with an external factor (e.g., a growth factor) to achieve pluripotency. In some aspects, the precursor cells can be totipotent (or pluripotent) stem cells; pluripotent stem cells (induced or non-induced); a pluripotent stem cell; oligopotent stem cells and unipotent stem cells. In some aspects, the precursor cells may be from an embryo, infant, child, or adult. In some aspects, the precursor cells may be somatic cells that have been treated to impart pluripotency thereto by genetic manipulation or protein/peptide processing.

As used herein, the term "cellular component" is a single gene, protein, gene expressing mRNA, and/or any other variable cellular component or protein activity, such as the degree of protein modification (e.g., phosphorylation), for example, which is typically measured by one of skill in the art in biological experiments (e.g., by microarray or immunohistochemistry). Significant findings related to the complex network of biochemical processes underlying living systems, common human diseases, as well as gene discovery and structural determination can now be attributed to the use of cellular component abundance data as part of research processes. Cellular component abundance data can help identify biomarkers, differentiate disease subtypes, and identify toxic mechanisms.

FGF signalling pathway activators: fibroblast Growth Factors (FGFs) are a family of growth factors involved in angiogenesis, wound healing and embryonic development. In some aspects, one skilled in the art will appreciate that any FGF can be used in combination with a protein from the Wnt signaling pathway. Exemplary FGF signaling pathway activators can include small molecule or protein FGF signaling pathway activators, FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, and combinations thereof. siRNA and/or shRNA associated with FGF signaling pathways can be used to target cellular components to activate these pathways. Suitable amounts and durations will be readily understood by those of ordinary skill in the art.

WNT signaling pathway activator: modulators/activators of Wnt signaling pathways may include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt 16. In some aspects, modulation of a pathway may be through the use of small molecule modulators or protein modulators that activate the above pathway or proteins that activate the above pathway. For example, small molecule modulators of the Wnt pathway include, but are not limited to, lithium chloride; 2-amino-4, 6-disubstituted pyrimidine (hetero) arylpyrimidines; IQ 1; QS 11; NSC 668036; DCA β -catenin; 2-amino-4- [3,4- (methylenedioxy) -benzyl-amino ] -6- (3-methoxyphenyl) pyrimidine. In some aspects, the extrinsic molecules may include small molecules, such as WAY-316606; SB-216763; or BIO (6-bromoindirubin-3' -oxime). In some aspects, siRNA and/or shRNA associated with Wnt and/or FGF signaling pathways can be used to target cellular components to activate these pathways. One skilled in the art will appreciate that target cellular components include, but are not limited to, SFRP proteins; GSK3, Dkk1, and FrzB. Other modulators include molecules or proteins that inhibit GSK3 that activate the Wnt signaling pathway. Exemplary GSK3 inhibitors may include, for example, Chiron/CHIR99021, which inhibits GSK3 β. One of ordinary skill in the art will recognize GSK3 inhibitors useful in practicing the disclosed methods. The WNT signaling pathway activator can be administered in an amount sufficient to practice the disclosed methods. Suitable amounts and durations will be readily understood by those of ordinary skill in the art.

BMP activating agent: exemplary BMP signaling pathway activators may be selected from BMP2, BMP4, BMP7, BMP9, small molecules that activate the BMP pathway, proteins that activate the BMP pathway, and may include the following: noggin, Dorsomorphin, LDN189, DMH-1, ventromophins, and combinations thereof.

Organoid technology is a developing area. In short, organoids are "organ-like tissues," or three-dimensional tissues having structural tissues similar to those of the corresponding native organ. Organoids can be derived from precursor cells such as embryonic stem cells or induced pluripotent stem cells. Organoids are typically cultured in vitro using time sequences of growth factor manipulations that mimic embryonic development of the organ tissue of interest-a process commonly referred to as directed differentiation of precursor cells. In general, organoids may contain differentiated cell types that are functional in many cases, for example, parietal cells capable of secreting acid. That is, at present, organoids described in the literature differ in scope from naturally occurring organ tissue. For example, the organoid may lack one or more other characteristics of the vasculature or native organ that the organoid may be intended to mimic. To date, organoids have not been recognized to have a developing hematopoietic system or to produce large numbers of immune cells. The present disclosure seeks to address one or more such needs in the art.

As described herein, the methods and systems use time series of growth factor manipulations to mimic embryonic development of tissue in culture, with modifications to allow development of immune system cells, variations of which are described herein.

Hematopoietic stem and progenitor cells

The production of blood cells is derived from a single type of cell, the hematopoietic stem cell, which, through proliferation and differentiation, produces the entire hematopoietic system. It is believed that hematopoietic stem cells are capable of self-renewal, expanding their own stem cell population, and that they are pluripotent (capable of differentiating into any cell in the hematopoietic system). From this rare population of cells, the complete adult hematopoietic system is formed, comprising lymphocytes (B and T cells of the immune system) and bone marrow cells (erythrocytes, megakaryocytes, granulocytes, and macrophages). Lymphoid lines comprising B cells and T cells contribute to the production of antibodies, regulation of the cellular immune system, detection of foreign substances in the blood, detection of host foreign cells, and the like. The myeloid lineage includes monocytes, granulocytes, megakaryocytes and other cells, monitors for the presence of foreign material, provides protection against tumor cells, eliminates foreign material, produces platelets, and the like. The erythroid cell line provides erythrocytes as oxygen carriers.

As noted above, current therapies to replace HSCs involve bone marrow transplantation. Because of the difficulties in "fitting" patients, there is a need in the art for compositions and methods that meet this need. To this end, organoid compositions and methods of making hematopoietic cell-producing organoids are disclosed herein.

The disclosed compositions and methods can be used to produce hematopoietic cells, which can be used to treat any disease state in which administration of hematopoietic cells is beneficial. Thus, the method may further comprise isolating or collecting hematopoietic cells from the disclosed organoid composition. Disease states that can be treated using the disclosed organoid-derived hematopoietic cells can include, for example, genetic diseases such as β -thalassemia, sickle cell anemia, adenosine deaminase deficiency, recombinase regulated gene deficiency by introducing a wild-type gene into stem cells, for example, using CRISPR techniques. In certain aspects, organoids and/or hematopoietic cells disclosed herein can be used to reconstitute a irradiated host or a host receiving chemotherapy.

Composition comprising a metal oxide and a metal oxide

Also disclosed herein are hematopoietic stem cell compositions, e.g., highly concentrated hematopoietic stem cell compositions, which are substantially free of differentiated or specialized hematopoietic cells. By "substantially free" is meant that less than 10%, or less than 5% or less than 1% of the cells are present in the population. The hematopoietic cells derived from the organoid composition may be a substantially homogenous composition of living mammalian or human hematopoietic cells, and may be produced for a variety of purposes, such as bone marrow transplantation, where the cells may be free of tumor cells or other pathogenic cells, such as HIV-infected cells, where transplantation of graft versus host disease is desired to be avoided. By "substantially homogeneous" is meant that the majority of cells in the composition are of the same cell type, e.g., at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or in some cases, greater than 95% of the desired cell type, wherein the cell type can be a hematopoietic stem cell. In certain aspects, hematopoietic cells can be modified by appropriate homologous or non-homologous recombination to correct genetic defects or to provide genetic capacity that is naturally absent in stem cells, whether for individuals or for stem cells in general. Such genetically modified cells (i.e., using CRISPR methods well known in the art) can be further administered to an individual in need thereof.

HCO culture producing immune cells

In a first aspect, a method of preparing a Hematopoietic Stem Cell (HSC) or cell derived therefrom is disclosed. The method may comprise contacting definitive endoderm derived from precursor cells with a wnt signaling pathway activator and an FGF signaling pathway activator until foregut cells are formed; and, culturing the foregut cells in the absence of retinoic acid to form a liver organoid that produces hematopoietic cells.

In one aspect, the precursor cells of any of the preceding paragraphs may be selected from one or both of embryonic stem cells and induced pluripotent stem cells (ipscs).

In one aspect, the Wnt signaling pathway activator of any one of the preceding paragraphs may be selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, small molecule activators of the Wnt signaling pathway (e.g., lithium chloride; 2-amino-4, 6-disubstituted pyrimidine (hetero) arylpyrimidine; IQ 1; QS 11; NSC 668036; DCA β -catenin; 2-amino-4- [3,4- (methylenedioxy) -benzyl-amino ] -6- (3-methoxyphenyl) pyrimidine), WAY-316606; SB-216763; or BIO (6-bromoindirubin-3' -oxime)), siRNA and/or shRNA activators of the Wnt signaling pathway, GSK3 inhibitors (e.g., Chiron/CHIR9902), and combinations thereof.

In one aspect, the FGF signaling pathway activator of any one of the preceding paragraphs may be selected from the group consisting of small molecule or protein FGF signaling pathway activators, FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, siRNA and/or shRNA activators of the FGF signaling pathway, and combinations thereof.

In one aspect, any of the preceding paragraphs may further comprise forming spheroids from the foregut cells prior to forming the liver organoids. In other aspects, the foregut cell may form a spheroid prior to forming the liver organoid, and the method may further comprise disrupting the spheroid to form a plurality of cells derived from the spheroid. Disruption may be accomplished by one or both of chemical disruption and/or mechanical disruption. For example, in one aspect, disruption may comprise treatment with an enzyme, e.g., an enzyme having one or both of proteolytic and collagenolytic activity, e.g., one or more enzymes selected from accutase, trypsin, collagenase, hyaluronidase, deoxyribonuclease, papain, trypzen (manufactured by Sigma), or a combination thereof.

In one aspect, any of the preceding paragraphs may further comprise culturing the foregut in the presence of a cytokine. The cytokine may be any cytokine acceptable in the art, for example, selected from the group consisting of transferrin, Stem Cell Factor (SCF), interleukin 3(IL-3), interleukin 6(IL-6), Erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), and combinations thereof. In certain aspects, the foregut may be dissociated into single cells prior to culturing. The culturing with the cytokine may be for a period of time, for example, about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days, or about 21 days, or about three weeks, or about four weeks, or about five weeks, or about six weeks, or about seven weeks, or about eight weeks, or about nine weeks, or about 10 weeks, or about 11 weeks, or about 12 weeks, or greater than 12 weeks.

In one aspect, the method of any one of the preceding paragraphs may further comprise contacting the liver organoid (e.g., a human liver organoid) with one or both of Thrombopoietin (TPO) and Stem Cell Factor (SCF), wherein the contacting with one or both of Thrombopoietin (TPO) and Stem Cell Factor (SCF) is for a period of time, e.g., selected from about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days, or about 21 days, or about three weeks, or about four weeks, or about five weeks, or about six weeks, or about seven weeks, or about eight weeks, or about nine weeks, or about 10 weeks or about 11 weeks, or a period of about 12 weeks, or greater than 12 weeks.

In one aspect, the liver organoid of any of the preceding paragraphs may be a liver organoid understood to be in a fetal state, e.g., a human liver organoid derived from human precursor cells, such organoids comprising fetal liver tissue. For example, in one aspect, the liver organoids may produce reduced albumin compared to human liver organoids treated with retinoic acid. In one aspect, the liver organoid produces alpha-fetoprotein (AFP). In one aspect, the liver organoids have increased endothelial markers CD34 and KDR compared to liver organoids treated with retinoic acid. In one aspect, the liver organoids have increased Erythropoietin (EPO) and hemoglobin gamma (HBG) compared to liver organoids treated with retinoic acid.

In one aspect, the method may comprise the steps of any of the preceding paragraphs, and further, suspending the foregut cells in a basement membrane matrix (e.g., Matrigel)^TM) In (1). The foregut cells may be further cultured on a stromal cell line (e.g., derived from bone marrow).

The method of claim 1, wherein the derivative cells are selected from the group consisting of bone marrow cells (such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes and platelet-producing megakaryocytes), lymphoid cells (such as T cells, B cells and natural killer cells) and combinations thereof.

HCO and HCO culture producing immune cells

In one aspect, a Human Colon Organoid (HCO) comprising a hematogenic endothelium and methods of making the same are disclosed. In one aspect, the hematogenic endothelium of an HCO described herein produces immune cells, such as one or more of erythrocyte-myeloid progenitor cells, lymphoid progenitor cells, and macrophages. In one aspect, the hematogenic endothelium of the disclosed HCO produces macrophages that secrete proinflammatory cytokines. The disclosed HCO may comprise a hematopoietic progenitor cell, wherein the progenitor cell is CD34+, wherein the CD34 progenitor cell is in organoid mesenchyme, wherein the hematopoietic progenitor cell has the capacity to form T-cells. In other aspects, the disclosed HCO may comprise endothelial tubes, wherein Endothelial Tubes (ET) are positive for CD34+, and wherein ET comprises RUNX1+ cells.

The colon organoids can be derived from precursor cells, such as human precursor cells, and can be used to obtain immune cells. In this aspect, a method comprises culturing a colon organoid to form an organoid culture; harvesting one or more immune cells from the colon organoid culture is also disclosed.

In this aspect, the colon organoid may be cultured for about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days, or about 21 days, or about three weeks, or about four weeks, or about five weeks, or about six weeks, or about seven weeks, or about eight weeks, or about nine weeks, or about 10 weeks, or about 11 weeks, or about 12 weeks, or more than 12 weeks, or until the colon organoid comprises one or more of hematopoietic endothelium and endothelial tubes that produce hematopoietic progenitor/stem cells.

In one aspect, the method may comprise isolating mesenchyme from the colon organoid culture and culturing the mesenchyme. In one aspect, the mesenchymal culturing step may last for a period of about four days to three months, or about five days to two months, or about 6 days to about one month, or about seven days to about 21 days. In another aspect, the mesenchymal culture may be a suspension culture.

In one aspect, the colon organoid may comprise mesenchyme, and wherein the culturing step may last for a period of time, for example, from about four days to three months, or from about five days to two months, or from about 6 days to about one month, or from about seven days to about 14 days. The culturing step may be in the form of suspension culture for a period of about one to four weeks, or about one week, to allow for expansion of the mesenchyme.

In one aspect, the immune cells of the disclosed methods can be selected from erythroid, myeloid, and mixed myeloid colonies. In other aspects, the immune cell can be one or more of a macrophage, neutrophil, eosinophil, basophil, erythrocyte, leukocyte, and monocyte.

In one aspect, a colon organoid can be derived from definitive endoderm derived from precursor cells described herein. In one aspect, the precursor cell is an embryonic stem cell or an induced pluripotent stem cell.

In one aspect, the method may further comprise culturing the organoid culture with a T cell inducible growth factor. In other aspects, the method may comprise disrupting the culture to disperse the colon organoids into individual colon organoids and disrupting the mesenchyme in the culture. This step may be performed by culturing the resulting disrupted organoids and mesenchyme in a basement membrane matrix (e.g., Matrigel) for a period of about one week to about four weeks, or about two weeks to about three weeks.

In other aspects, colon organoids, particularly human colon organoids, can be used to model disease states. For example, a method of modeling a disease state selected from necrotizing enterocolitis, very early-onset IBD30, infection from bacterial pathogens (e.g., clostridium difficile), infection from viral pathogens (e.g., HIV, which is prone to infect fetal intestinal macrophages) is disclosed. In this aspect, the method may comprise inducing a disease state in a colon organoid (e.g., a human colon organoid) prepared according to the methods disclosed herein.

In one aspect, a method of making a HCO or HIO capable of producing Hematopoietic Stem Cells (HSCs) is disclosed, wherein the method can comprise contacting definitive endoderm derived from precursor cells with one or more factors for a sufficient period of time to produce mid/posterior intestinal spheroids, optionally embedding the mid/posterior intestinal spheroids in a basement membrane matrix, and contacting the DE with a combination of factors comprising FGF, CHIR, noggin, and a SMAD inhibitor, in an amount and for a sufficient time to produce anterior foregut forespheres; wherein the mid/posterior intestinal spheroids or anterior foregut spheroids produce HSCs.

In another aspect, a method of treating an individual in need of immune cells is disclosed. The method may comprise harvesting Hematopoietic Stem Cells (HSCs) or derived cells from HCO or HLO according to any one of the preceding paragraphs; and administering the HSCs or their derived cells to an individual in need thereof, wherein the administering comprises transplanting the HSCs into the bone marrow of the individual. In one aspect, the treatment can be a treatment for anemia (including aplastic anemia, fanconi anemia), immunodeficiency, cancer (e.g., lymphoma, leukemia, cancer, solid tumor), hematopoietic genetic disorders, genetic storage disease, thalassemia major, sickle cell disease, osteoporosis, or a combination thereof.

Precursor cell

In some aspects, pluripotent stem cells may be used or may be induced to become pluripotent. In some aspects, pluripotent stem cells are derived from embryonic stem cells, which in turn are derived from totipotent cells of early mammalian embryos and are capable of unlimited, undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of a blastocyst (i.e., early embryo). Methods for deriving embryonic stem cells from embryonic cells are well known in the art. For example, three cell lines (H1, H13 and H14) have a normal XY karyotype and two cell lines (H7 and H9) have a normal XX karyotype. Human embryonic stem cell H9(H9-hESC) is used in the exemplary aspects described herein, but it will be understood by those skilled in the art that the methods and systems described herein are applicable to any stem cell. Additional stem cells that may be used in accordance with aspects of the invention include, but are not limited to, those provided by or described in a database hosted by: national Stem Cell Bank (NSCB), University of California, San Francisco, Human Embryonic Stem Cell Research Center (Human Embryonic Stem Cell Research Center at the University of California, San Francisco, UCSF); WiSC Cell Bank at Wi Cell Research Institute; university of Wisconsin Stem cells and Regenerative Medicine Center (the University of Wisconsin Stem Cell and Regenerative Medicine Center, UW-SCRMC); norwessel (Novocell, Inc.) (san diego, california); cellartis AB (gold burger, sweden); ES Cell International Pte Ltd (singapore); israel Institute of Technology (Technology at the Israel Institute of Technology, Israel sea); and a stem cell database hosted by university of princeton and university of pennsylvania. Exemplary embryonic stem cells that can be used in accordance with aspects of the invention include, but are not limited to, SA01(SA 001); SA02(SA 002); ES01 (HES-1); ES02 (HES-2); ES03 (HES-3); ES04 (HES-4); ES05 (HES-5); ES06 (HES-6); BG01 (BGN-01); BG02 (BGN-02); BG03 (BGN-03); TE03 (I3); TE04 (I4); TE06 (I6); UC01(HSF 1); UC06(HSF 6); WA01 (H1); WA07 (H7); WA09 (H9); WA13 (H13); WA14 (H14). In some aspects, the stem cells are further modified to introduce additional properties. Exemplary modified cell lines include, but are not limited to, H1 OCT 4-EGFP; h9 Cre-LoxP; h9 hNanog-pGZ; h9 hOct 4-pGZ; h9 inGFPheS; and H9 Syn-GFP. More details on Embryonic Stem cells can be found, for example, in Thomson et al, 1998, "Embryonic Stem Cell Lines Derived from Human Blastocysts (Embryonic Stem cells Lines Derived from Human blast combustors" [ Science 282 (5391)): 1145-; andrews et al, 2005, "Embryonic Stem (ES) cells and Embryonic Carcinoma (EC) cells: opposite sides of the same coin (Embryonic stem (ES) cells and Embroyal Carcinoma (EC) cells: open sites of the same coin), Biochem Soc Trans 33: 1526 and 1530; martin 1980, in Teratocarcinomas and mammalian embryogenesis, Science 209 (4458): 768-776; evans and Kaufman, 1981, "Establishment of mouse embryo pluripotent cell cultures (obtaining in culture of pluripotent cells from mouse embryos"), Nature 292 (5819): 154-156; klimanskaya et al, 2005, "feeder cells-free derived Human embryonic stem cells (Human embryonic cells derived with feeder cells)," lancets (Lancet) 365 (9471): 1636-1641; each of which is incorporated herein in its entirety. Alternatively, pluripotent stem cells may be derived from Embryonic Germ Cells (EGCs), which are cells that produce gametes of sexually reproducing organisms. EGCs are derived from primordial germ cells found in the gonadal ridges of late-stage embryos and possess many of the characteristics of embryonic stem cells. Primordial germ cells in the embryo develop into stem cells, producing germ gametes (sperm or ova) in the adult. In mice and humans, embryonic germ cells can be grown in tissue culture under suitable conditions. Both EGC and ESC are versatile. For the purposes of the present invention, the term "ESC" is sometimes used broadly to encompass EGCs.

Induced Pluripotent Stem Cells (iPSC)

In some aspects, ipscs are derived by transfecting certain stem cell-associated genes into non-pluripotent cells (e.g., adult fibroblasts). Transfection is accomplished by viral vectors (e.g., retroviruses). The transfected genes included the major transcriptional regulators Oct-3/4(Pouf51) and Sox2, although other genes were suggested to enhance induction efficiency. After 3-4 weeks, a small number of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells and are usually isolated by morphological selection, doubling time or by reporter gene and antibiotic selection. As used herein, ipscs include, but are not limited to, mouse first generation ipscs, second generation ipscs, and human induced pluripotent stem cells. In some aspects, ipscs can be generated using non-virus based techniques. In some aspects, the adenovirus can be used to transport the necessary four genes into the DNA of mouse skin and hepatocytes, resulting in the same cells as embryonic stem cells. Since adenovirus does not bind any of its own genes to the target host, the risk of developing tumors is eliminated. In some aspects, reprogramming can be accomplished by plasmids without any viral transfection system at all, although at very low efficiency. In other aspects, direct delivery of the protein is used to generate ipscs, thus eliminating the need for viral or genetic modifications. In some embodiments, it is possible to generate mouse ipscs using a similar approach: repeated treatment of cells with certain proteins introduced into the cells via poly-arginine anchors is sufficient to induce pluripotency. In some aspects, expression of pluripotency-inducing genes can also be increased by treating somatic cells with FGF2 under hypoxic conditions. More details on embryonic stem cells can be found, for example, in Kaji et al, 2009, Virus free induction of pluripotency and subsequent ablation of reprogramming factors (Virus free induction of pluripotency and subsequent exposure of reprogramming factors), Nature 458: 771-775; woltjen et al, 2009, "reprogramming fibroblasts into induced pluripotent stem cells by transposition," Nature 458: 766-770; okita et al, 2008, Generation of Virus vector-free mice induced pluripotent stem cells with out viral vectors, Science 322 (5903): 949-; stadtfeld et al, 2008, "Induced pluripotent stem cells without Viral integration" (Science) 322 (5903): 945 949; and Zhou et al, 2009, "Generation of induced pluripotent Stem cells (induced pluripotent Stem cells using recombinant proteins" ") Cell Stem cells (Cell Stem cells)" 4 (5): 381-384; each of which is incorporated herein in its entirety. In certain aspects, exemplary iPS cell lines include, but are not limited to, iPS-DF 19-9; iPS-DF 19-9; iPS-DF 4-3; iPS-DF 6-9; iPS (foreskin); iPS (IMR 90); and iPS (IMR 90).

Definitive endoderm. Spheroids, organoids, and/or tissues described herein may be derived from a monolayer of cells known as Definitive Endoderm (DE). Methods for deriving definitive endoderm from precursor cells are well known in the art, as taught by D' Armour et al 2005 and Spence et al. Any method of producing definitive endoderm from pluripotent cells (e.g., iPSCs or ESCs) is suitable for use in the methods described herein. In some aspects, the pluripotent cells are derived from morula. In some aspects, the pluripotent stem cells are stem cells. The stem cells used in these methods may include, but are not limited to, embryonic stem cells. Embryonic stem cells may be derived from the inner cell mass of an embryo or the gonadal ridges of an embryo. Embryonic stem or germ cells can be derived from various animal species, including but not limited to various mammalian species, including humans. In some aspects, human embryonic stem cells are used to produce definitive endoderm. In some aspects, human embryonic germ cells are used to produce definitive endoderm. In certain aspects, ipscs are used to produce definitive endoderm. In some aspects, one or more growth factors are used in the differentiation process from pluripotent stem cells to DE cells. The one or more growth factors used in the differentiation process may include growth factors from the TGF- β superfamily. In these aspects, the one or more growth factors may comprise Nodal/activin and/or a subgroup of BMPs of the TGF- β superfamily. In some aspects, the one or more growth factors are selected from the group consisting of: nodal, activin a, activin B, BMP4, Wnt3a, or a combination of any of these growth factors. In some aspects, the embryonic stem cells or germ cells and ipscs are treated with one or more growth factors for 6 hours or more; 12 hours or more; 18 hours or more; 24 hours or more; 36 hours or more; 48 hours or more; 60 hours or more; 72 hours or more; 84 hours or more; 96 hours or more; 120 hours or more; 150 hours or more; 180 hours or more; or 240 hours or more. In some aspects, embryonic stem or germ cells and ipscs are treated with one or more growth factors at the following concentrations: 10ng/ml or higher; 20ng/ml or higher; 50ng/ml or higher; 75ng/ml or higher; 100ng/ml or more; 120ng/ml or higher; 150ng/ml or higher; 200ng/ml or higher; 500ng/ml or more; 1,000ng/ml or more; 1,200ng/ml or more; 1,500ng/ml or more; 2,000ng/ml or more; 5,000ng/ml or more; 7,000ng/ml or more; 10,000ng/ml or more; or 15,000ng/ml or higher. In some aspects, the concentration of the growth factor is maintained at a constant level throughout the treatment. In other aspects, the concentration of the growth factor is varied during the treatment. In some aspects, the growth factor is suspended in a medium comprising Fetal Bovine Serine (FBS) and different fetal bovine serum concentrations. One skilled in the art will appreciate that the protocols described herein can be applied to any known growth factor, alone or in combination. When two or more growth factors are used, the concentration of each growth factor may be independently varied. In some aspects, a cell population enriched for definitive endoderm cells is used. In some aspects, definitive endoderm cells are isolated or substantially purified. In some aspects, isolated or substantially purified definitive endoderm cells express SOX17, FOXA2, and/or CXRC4 markers to a greater extent than OCT4, AFP, TM, SPARC, and/or SOX7 markers. Methods of enriching a population of cells having definitive endoderm are also contemplated. In some aspects, definitive endoderm cells can be isolated or substantially purified from a mixed population of cells by contacting the cells with an agent that binds to a molecule present on the surface of the definitive endoderm cells but not on the surface of other cells in the mixed population of cells, and then isolating the cells bound to the agent. In certain aspects, the cellular component present on the surface of definitive endoderm cells is CXCR 4. Other methods of obtaining or producing DE cells that may be used in the present invention include, but are not limited to, those described in the following references: U.S. Pat. Nos. 7,510,876 to D' Amour et al; fisk et al, U.S. Pat. Nos. 7,326,572; kubo1 et al, 2004, "Development of definitive endoderm (Development) 131 in embryonic stem cells during culture: 1651-1662; d' Amour et al, 2005, "effective differentiation of human embryonic stem cells to definitive endoderm," Nature Biotechnology (Nature Biotechnology) "23: 1534-1541; and Ang et al, 1993, formation and maintenance of mouse malformed endoderm layers: involvement of HNF3/forkhead proteins (The formation and main of The expression end linkage in The mouse: nonvolatile of HNF3/forkhead proteins) '(Development)' (119): 1301-1315; each of which is incorporated herein by reference in its entirety. In some aspects, soluble FGF and Wnt ligands are used to mimic early hindgut specification in culture, transforming DE developed from ipscs or ESCs into the hindgut epithelium that efficiently produces all major intestinal cell types by directed differentiation. In humans, directed differentiation of DE is achieved by selective activation of certain signaling pathways important for intestinal development. It will be appreciated by those skilled in the art that altering the expression of any Wnt signaling protein in combination with any FGF ligand may result in directed differentiation as described herein. In some aspects, DE cultures are treated with one or more modulators of the signaling pathway described herein for 6 hours or more; 12 hours or more; 18 hours or more; 24 hours or more; 36 hours or more; 48 hours or more; 60 hours or more; 72 hours or more; 84 hours or more; 96 hours or more; 120 hours or more; 150 hours or more; 180 hours or more; 200 hours or more; 240 hours or more; 270 hours or more; 300 hours or more; 350 hours or more; 400 hours or more; 500 hours or more; 600 hours or more; 700 hours or more; 800 hours or more; 900 hours or more; 1,000 hours or longer; 1,200 hours or longer; or 1,500 hours or more.

In some aspects, DE cultures are treated with one or more modulators of the signaling pathway described herein at the following concentrations: 10ng/ml or higher; 20ng/ml or higher; 50ng/ml or higher; 75ng/ml or higher; 100ng/ml or more; 120ng/ml or higher; 150ng/ml or higher; 200ng/ml or higher; 500ng/ml or more; 1,000ng/ml or more; 1,200ng/ml or more; 1,500ng/ml or more; 2,000ng/ml or more; 5,000ng/ml or more; 7,000ng/ml or more; 10,000ng/ml or more; or 15,000ng/ml or higher. In some aspects, the concentration of the signaling molecule remains constant throughout the treatment. In other aspects, the concentration of the signaling pathway modulator is varied during the treatment. In some aspects, the signaling molecule according to the invention is suspended in a medium comprising DMEM and Fetal Bovine Serine (FBS). The concentration of FBS may be 2% or higher; 5% and higher; 10% or more; 15% or more; 20% or more; 30% or more; or 50% or higher. One skilled in the art will appreciate that the protocols described herein may be applied to any known modulator of the signaling pathway described herein, including but not limited to, any molecule in the Wnt and FGF signaling pathways, alone or in combination.

In aspects where two or more signaling molecules are used to treat the DE culture, the signaling molecules may be added simultaneously or separately. When two or more molecules are used, the concentration of each molecule can be independently varied.

Examples of the invention

The following non-limiting examples are provided to further illustrate the embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent methodologies which have been found to function well in the practice of the invention, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Establishment of functional immune cells in colon organoid cultures derived from human pluripotent stem cells

Committed hematopoietic progenitor cells arise from the hematopoietic endothelium that develops near the embryonic colon. Here, applicants have designed colon organoid cultures derived from human pluripotent stem cells that together develop hematopoietic endothelial and progenitor cells competent to form myeloid and lymphoid derivatives. BMP signaling can be used to generate hindgut mesenchyme, which is capable of forming hematogenic endothelium and producing hematopoietic progenitor cells expressing RUNX 1. After three weeks of culture, there are a variety of bone marrow cell types and lymphocytes. Following expanded in vitro culture, macrophages can be maintained in the developing mesenchyme of HCO. PSC-derived human macrophages establish tight association with colonic epithelium after HCO transplantation and 3 months of in vivo growth and are not replaced by host-derived macrophages. HCO-associated macrophages are functional and respond to LPS and pathogenic bacteria by producing inflammatory cytokines, undergoing transepithelial migration and phagocytosis of bacteria (all characteristic of tissue resident macrophages). As in the embryo, applicants have designed human hindgut/colon organoid cultures that support the formation of hematogenic endothelium that produces bone marrow and lymphoid lineages, including the establishment of long-term resident macrophages in the developing human colon.

There are a wide variety of immune cells distributed in the adult gut. These include myeloid and lymphoid cell types that coordinate with epithelium and ENS to maintain barrier function, communicate with microbiome, and differentiateBeneficial and harmful antigens. Most intestinal diseases, particularly Inflammatory Bowel Disease (IBD), involve the immune system. The dogma states that all immune cells of the gut are derived from bone marrow derived Hematopoietic Stem Cells (HSCs). However, increasing evidence from animal studies supports the conclusion that certain organs contain tissue resident macrophage populations that develop together during embryonic development^1-3. It has recently been shown that the colon contains a stable, self-sustaining population of macrophages derived from embryonic progenitor cells and adult HSCs⁴。

Hematopoietic cells develop from three sites. Primitive hematopoietic cells are produced during gastrulation, migrate to the yolk sac and survive shortly⁵. Shaped Hematopoietic Progenitor Cells (HPCs) are derived from the blood-producing endothelium, located in the yolk sac or in the aortic-gonadal-mesorenal (AGM) region of the embryo, adjacent to the developing colon. One of the notable features of primitive hematopoietic cells is that they have limited differentiation potential and do not have lymphoid potential. The in-embryo HPC derived from the AGM region expresses Runx 1-and Tek-like markers and emerges from the aortic endothelium and the peripheral blood vessels adjacent to the hindgut⁶. BMP signalling is required for the development of the posterior region of the embryo^7-10. In addition, BMP signaling regulates the expression of the GATA2 transcription factor required for hematogenous endothelialization¹¹。

Applicants have previously completed the design of Human Colon Organoids (HCO) by directed differentiation of human pluripotent stem cells¹⁰. Such HCOs contain colonic epithelial and peripheral mesenchymal derivatives, including fibroblasts, myofibroblasts and smooth muscle cells. Bioinformatic analysis of the transcriptional changes that occur at the HCO differentiation stage revealed a surprising enrichment of genes associated with hematopoietic development. Disclosed herein are methods of treating a culture to identify the extent of cell types present in HCO, as shown in figure 10.

Hematopoietic progenitor cells develop from the caudal mesoderm. In order to determine whether caudal mesoderm has been designated, applicants examined the expression of the HOX gene expressed forward and backward. Consistent with previous findings, the post hox gene was significantly upregulated by BMP treatment (fig. 11, panel D). Furthermore, pro-hox factors, including HOXA3, that inhibit EHT12 were down-regulated in HCO compared to HIO. When the development of hindgut and colon fates was induced by transient activation of BMP signaling (BMP2 treatment occurred between days 7-10 of the protocol), expression of vascular markers, including GATA2, KDR/FLK1, as well as the whole endothelial markers CD34 and VEGFR1, was observed, similar to that observed in the hindgut of mouse embryonic development (figure 11).

The presence of immune cells in HCO culture raises the question of whether these cells are primitive or derived from hematogenic endothelium. No expression of GYPA (CD235) labeling primitive hematopoietic progenitor cells was observed (FIG. 11, F)¹³Indicating that the source of hematopoietic cells is hematopoietic endothelium. Hematopoietic progenitor cells expressing RUNX1 were seen to emerge from the endothelium of the AGM region of e10.5 mouse embryos (fig. 4 (a-C)). HCO cultures similarly had CD34+ endothelial tubes and associated RUNX1+ cell clusters (fig. 4 (D-G)). The hematopoietic endothelium also differs from non-hematopoietic endothelium by the lack of CD73 expression¹⁴. Analysis of 21 day old HCO cultures by flow cytometry revealed the presence of CD34⁺CD73 endothelial cells, indicating the presence of a hematopoietic endothelium (FIG. 4 (H)). By 21 days in culture, the transcriptional pattern of HCO compared to HIO revealed pathway terminology associated with immune cells and their function. These include neutrophil degranulation, innate immune system, platelet activation and leukocyte transendothelial migration. Analysis of 21 day old HCO cultures by immunofluorescence staining (IF) confirmed the presence of PU.1⁺Cells and CD34⁺Endothelial tubes embedded in the mesenchyme of colon organoids, but not intestinal organoids (fig. 12). Bright field in vivo imaging of 22 day old HCO culture cultures revealed refractory cells migrating in the endothelial cell tube, which appeared free floating in the mesenchyme and in the culture medium and could be found. Taken together, these data indicate that HCO cultures contain a hematopoietic endothelium capable of producing hematopoietic cells.

To determine the cell type in HCO cultures, media was sampled and cytospin (cytospins) and giemsa stained, which identified cells like macrophages, neutrophils, eosinophils and basophils (fig. 5A). No exogenous factors or mouse bone marrow stromal cells were added to the cultures, indicating HCOCan support differentiation of bone marrow cell types. To determine whether HCO-producing erythroid myeloid progenitor cells were present, MethoCult was performed^TMAnd (6) analyzing. HCO, but not HIO cultures, contained progenitor cells capable of producing erythroid, bone marrow and mixed bone marrow colonies (fig. 5, B). HCO from human embryos and induced pluripotent stem cell lines has the ability to generate erythroid myeloid derivatives, demonstrating that the method is effective in PSC lines. Erythrocytes produced from HCO express fetal (HBG1 and 2) and fetal/adult (HBA1, HBA2) hemoglobin but do not express appreciable levels of embryonic hemoglobin (HBE1, HBZ), indicating that HCO cultures contain shaped erythroid myeloid progenitor cells.

Table of human hemoglobin isoforms during development

One of the hallmarks of the formation of hematopoietic cells in the embryo is the ability to form lymphoid cell types, such as T cells^13,15. Whereas hematopoietic progenitors with lymphoid potential appear at a later stage of embryonic development, applicants postulate that these progenitors appear after prolonged culture of HCO mesenchyme. Thus, the applicant developed a culture method that allows the long-term maintenance of intact hematogenic endothelium (fig. 10, C). HCO cultures were grown for an additional week to allow for expansion of the mesenchyme, scraped from the plate and grown in suspension culture for an additional 3 weeks. To test for lymphoid potency, T cell inducible growth factors IL7 and FLT3 were added. Without T-cell induction, HCO cultures contained 0.2% CD3+/CD4+ cells. Addition of T-cell inducing growth factors increased the number of T-cells 4-fold (FIG. 5, E). The presence of T cell potential further supports the definitive conclusion that hematopoietic cells are formed from HCO cultures.

When the stereogenic function is transferred to other organs during development,including fetal liver, then bone marrow, tissue resident macrophages are able to grow in organs early in development and persist to birth. In some organs, such as the lung and liver, embryonic macrophages persist throughout life 5. In other organs, HSCs in postnatal bone marrow produce macrophages that replace embryonic macrophages. In the colon, several data indicate that embryonic macrophages are replaced by HSC-derived macrophages^16,17. However, recent lineage tracing of embryonic macrophages indicates that they persist with HSC-derived macrophages after birth. Colon organoids were passaged by abrasion on day 21, which resulted in disruption of the mesenchyme and dispersion of individual HCOs. The HCO was then re-spread in Matrigel and incubated for an additional 14 days. When the transcriptional pattern was detected by gene ontology analysis, an enrichment of the GO term associated with myeloid cell types including leukocytes, neutrophils, and defense and inflammatory responses was observed.

TABLE 35 days HCO Up-Regulation

Categories	ID	Name (R)	P value
				GO cellular Components	GO:0005615	Extracellular space	8.76E-26
GO cellular Components	GO:0031226	Intrinsic constituents of plasma membranes	S.53E-19
				GO-bioprocess	GO:0006954	Inflammatory reaction	4.79E-18
GO-bioprocess	GO:0006952	Defense reaction	3.13E-17
				GO cellular Components	GO:0005887	Integral component of plasma membrane	9.87E-17
GO-bioprocess	GO:1903034	Modulation of nociceptive response	3.01E-16
				GO-bioprocess	GO:0009611	Reaction to injury	1.07E-15
GO-bioprocess	GO:0030198	Extracellular matrix tissue	5.09E-15
				GO-bioprocess	GO:0043062	Extracellular structural tissue	5.78E-15
GO cellular Components	GO:0098589	Film zone	5.48E-13
				GO cellular Components	GO:0045177	Cell tip portion	4.32E-12

Immunostaining of HCO at day 35 revealed macrophage expression markers CD68 and HAM56 (data not shown) and tissue resident macrophage marker CD163 (fig. 12)^18,19. CD163 is expressed in several tissue-resident macrophage populations, including alveolar macrophages, Kupffer cells in the liver and Hofbauer cells in the placenta^20-22. The presence of inflammatory macrophages was studied by co-staining CD163 and iNOS, a known marker of inflammatory macrophages (fig. 7). Interestingly, most CD163+ macrophages were also positive for iNOS, indicating that these cells are inflammatory. Taken together, these data indicate that HCO contains co-developing macrophages co-expressing tissue resident and inflammatory macrophage markers.

The human intestine is composed of multiple macrophage subtypes. To determine whether macrophages are heterogeneous in HCO, CYTOF was used to detect expression of cell surface markers. CYTOF analysis revealed the presence of at least 4 different monocyte populations including the CD11bhi population, the CD14-/CD16+ population, the CD14+/CD16+ population and the CD14+/CD 16-population. These data indicate that HCO cultures are able to produce a different set of monocytes/macrophages resembling the native human intestine.

Groups in the colon have been postulatedTissue resident macrophages are continuously replenished by Bone Marrow Derived Monocytes (BMDM)^16,17. However, recent studies challenge this paradigm and suggest that while some macrophage subtypes continue to be supplemented by BMDM, other subtypes have a long-term survival and self-sustaining embryonic origin^4,23,24. To determine whether HCO macrophages ("HCOMac") could be maintained long term, the presence of human CD163+ macrophages was examined after HCO transplantation into the mouse kidney capsule (fig. 8). Applicants hypothesized that short lived macrophages would be replaced by host-derived murine macrophages expressing the mouse specific marker F/480. Only a few hCD163+ cells were detected in the control, but not in the NOG HIO grafts, and F/480+ macrophages infiltrated all mesenchymal layers up to the top of the villus (fig. 5). In contrast, hCD163 macrophages were readily detectable in HCO even 12 weeks after transplantation. These macrophages are located primarily in the lamina propria lacking F/480+ macrophage infiltration. In the muscle layer, hCD163+ cells were interspersed with F/480+, indicating that host macrophages are growing in these tissue layers. Examination of blood and bone marrow of HCO-transplanted mice revealed a lack of human-derived cells, indicating that hCD163 macrophages are self-sustained in HCO and are not replenished by human cells growing in bone marrow (figure 14). These data indicate that HCO produces self-sustained macrophages independent of BMDM.

Examination of RNAseq data from 35 day HCO revealed inflammatory features compared to HIO. To confirm that HCO did exhibit functional inflammation, Luminex multiplex ELISA was used to detect proinflammatory cytokine secretion into HCO medium (fig. 13). IL1B, IL6 and IL8 were all reported to be expressed in vitro by epithelial cells, suggesting that epithelial cells may contribute to the inflammatory features seen in HCO. To ensure that macrophages are inflammatory, secretion of macrophage inflammatory proteins 1A (MIP1A) and 1B (MIP1B) was examined. HCO secreted higher levels of MIP1A and MIP1B, indicating that macrophages within HCO showed basal levels of inflammation.

Adult colonic macrophages are generally resistant to stimulation by Lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria²⁵. In contrast, fetal macrophages respond to LPS stimulation, indicating that tolerance is achieved postnatallyTo be received²⁶. To determine whether macrophages within HCO are susceptible to LPS stimulation, HCO was treated with LPS and cell motility and inflammatory cytokine secretion were measured. In vivo imaging revealed that macrophages increased their motility in response to LPS and that they were able to undergo chemotaxis to lesions within organoids (fig. 8 (a)). Examination of cytokine secretion revealed that IL6, IL8, MIP1A, MIP1B, and TNFA were significantly increased, indicating that cytokine production may be a driver of macrophage motility (fig. 8 (B-E)).

Direct stimulation of HCO by LPS indicates that macrophages within organoids are able to respond to cytokines and bacterial factors. Macrophages play a direct role in innate immunity by phagocytosing bacteria. To determine whether HCO macrophages ("HCOmac") are able to phagocytose bacteria, HCO was treated with e. In vivo imaging revealed that HCOMac continued to extend the filopodia and observe the microenvironment. HCOMac phagocytoses bacterial particles within the acidic phagolysosome, as indicated by an increase in fluorescence that is pH sensitive (FIG. 8 (F-G)). In addition, microinjection of live commensal E.coli and EHEC induced macrophage migration into the lumen of HCO (FIGS. 9(A-F)), similar to that observed in mice infected with Salmonella²⁷. Furthermore, introduction of bacteria into the cavity of HCO resulted in reduced staining of MUC2, possibly due to degradation of mucus by the bacteria (fig. 9 (G)). In summary, our data indicate that HCOMac is a functional resident macrophage capable of responding to bacterial particles and live bacteria.

The development of the hindgut and aorto-gonadal-mesorenal regions of mammals are very close to each other. BMP signaling was shown to activate both the post-HOX pattern of the post-intestinal germ layer and mesenchyme and the expression of the hematogenic endothelial transcription factor GATA 2. Using the previously described methods of HCO generation, BMP signaling also specifies a hematogenic endothelium with a defined hematopoietic potential. This is consistent with normal human development, with established hematopoietic progenitor cells formed from retroperitoneal mesoderm. Thus, it is believed that the HCO cultures described herein more closely mimic a larger portion of the posterior embryo than originally thought.

Hematopoietic progenitor cells from HCO cultures even inHematopoietic growth factors and mouse-derived bone marrow stromal cells (OP9-DLL4 cells) were also lacking with erythro-myeloid and lymphoid potential. This indicates that the co-developing mesoderm compensates for these signals and the lack of cell types in HCO culture. Interestingly, hematopoietic growth factors were expressed in HIO cultures, indicating that the development of cells expressing these factors is independent of BMP signaling. These cell types can be used instead of murine OP9-DLL4 cells and thus may confer immunogenicity on human hematopoietic progenitor cells^28,29. Furthermore, these cell types may also be present in normal intestinal tissue, as recent studies have shown that a subpopulation of macrophages is self-sustaining in the intestine⁴。

The presence of co-developing macrophages in HCO provides a new tool for detecting the interaction between innate immune cells and colonic epithelium. In addition, HCO can be used to determine niche factors that allow for maintenance of tissue resident macrophages. The HCO should allow modeling of inflammatory diseases, such as necrotizing enterocolitis, very early-onset IBD30, bacterial pathogens, such as clostridium difficile, viral pathogens, such as HIV, which readily infect fetal intestinal macrophages³¹. In addition, incorporation of other immune cell types can be used to study other innate immune mechanisms, such as neutrophil-driven inflammatory hypoxia³²。

Method

And (5) DE induction. Human ES and iPS cells were plated as single cells on mTesR1 medium, supplemented with ROCK inhibitor Y27632 (10. mu.M; Stemgent), placed in matrigel (BD biosciences) coated 24-well plates, 150,000 cells per well. Starting the next day, cells were treated with activin A (100ng ml-1; Cell Guidance Systems) in RPMI 1640(Invitrogen) for three days with increasing concentrations (0%, 0.2% and 2.0%) of shaped fetal bovine serum (dFBS; Invitrogen). Endodermal patterning and gut morphogenesis. After DE induction, cells were treated with growth factor/antagonist in RPMI 1640 with 2.0% dfs for three days. To generate posterior foregut spheroids, DE was treated with the following substances for 4 days: FGF4(500ng ml-1)；R&D Systems), CHIR99021(3 μ M; stemgent). And (4) three-dimensional growth. As described previously^10,12Mid-hindgut spheroids were embedded in matrigel (BD biosciences) and subsequently grown in Advanced DMEM/F12(Invitrogen) supplemented with N2(Invitrogen), B27(Invitrogen), L-glutamine, 10 μ M HEPES, penicillin/streptomycin and EGF (100ng ml-1; R&D Systems). For proximal intestinal specialization Noggin (100ng ml-1; R) was added&D Systems), added on the first three days of three-dimensional growth. For colon specialization BMP2(100ng ml-1; R) was added&D Systems), added on the first three days of three-dimensional growth.

Methods of generating human gut organoids (HIOs) with increased immune cell production

Human embryonic stem cells and induced pluripotent stem cells were maintained on matrigel (bd biosciences) in feeder cells-free mTesR1 medium. Differentiation into definitive endoderm was performed as described previously (D' AmourKA et al, effective differentiation of human embryonic stem cells into definitive endoderm) (Nature Biotechnol 2005; 23: 1534. briefly 1541.) using a 3-day activin A (R & D Systems) differentiation protocol. Cells were treated with activin a (100ng/mL) for three consecutive days in RPMI 1640 medium (Invitrogen) containing increasing concentrations (0%, 0.2%, 2%) of HyClone-shaped fetal bovine serum (dfs) (Thermo Scientific). For hindgut differentiation DE cells were cultured for up to 4 days in 2% dFBS-DMEM/F12 containing 500ng/ml FGF 4and 500ng/ml Wnt3a (R & D Systems). Between 2-4 days of treatment with growth factors, 3-dimensional floating spheroids were formed and then transferred to three-dimensional cultures previously shown to promote intestinal growth and differentiation (Gracz AD, Ramalingam S, Magnetess ST.. Sox9-Expression Marks a Subset of Small intestinal Epithelial Stem Cells expressing CD24 that Form Organoids in vitro (Sox9-Expression Marks a Subset of CD 24-Expression Small intestinal Epithelial Stem Cells expressing CD24 in vitro) (Sox9-Expression Marks a subunit of CD 24-Expression Small intestinal Epithelial Stem Cells in vitro) (Am J physical tissue light physical organ physical P. 2010; G590-600; 16.Sato T, et al. Single Lgr Stem Cells construct a crypt villus structure in vitro without a Single Lgr mesenchymal Stem cell type-pressed spheroid (Nature 1. Sprint) embedded in vitro (Nature 39265. spz AD) in vitro), 100ng/mL noggin (R & D Systems) and 50ng/mL EGF (R & D Systems) in matrigel (BD bioscience). After solidification of Matrigel, the medium was overlaid and replaced every 4 days (supplemented with L-glutamine, 10. mu.M Hepes, N2 supplement (R & D Systems), B27 supplement (Invitrogen) and Pen/Strep containing growth factors Advanced DMEM/F12 (Invitrogen)).

Method of generating Human Colon Organoids (HCO) with increased immune cell production

Human embryonic stem cells and induced pluripotent stem cells were grown in feeder-free conditions in six-well Nunclon surface plates (Nunc) coated with matrigel (bd biosciences) and maintained in mTESR1 medium (stem cell technology). To induce Definitive Endoderm (DE), human ES or iPS cells were passaged with accutase (invitrogen) and seeded at a density of 100,000 cells per well in Matrigel-coated Nunclon surface 24-well plates. For Accutase lysed cells, 10 μ M Y27632 compound (Sigma) was added to the medium the first day. After the first day, the medium was changed to mTESR1 and the cells were grown for a further 24 hours. Cells were then treated with 100ng/mL activin A for 3 days as previously described (Spence et al, 2011). After induction of DE, the DE was treated with hindgut induction medium (RPMI 1640, 2mM L-glutamine, 2% deconstituent FBS, penicillin-streptomycin), FGF4(R & D) 500ng/mL and Chiron 99021(Tocris, WNT pathway activator; inhibition GSK3) 3 μ M for 4 days to induce mid-hindgut spheroid formation. Intestinal spheroids were collected/retroenteric from 24-well plates and plated in matrigel (BD) as described previously, followed by growth in Advanced DMEM/F12(Invitrogen) supplemented with N2(Invitrogen), B27(Invitrogen), L-glutamine, 10 μ M HEPES, penicillin/streptomycin and EGF (100ng ml-1; R & D Systems). To generate Human Colon Organoids (HCO), spheroids were covered with 100ng/mL EGF plus 100ng/mL BMP (BMP2 or 4, R & D or other BMP pathway activators may also be used) for at least 3 days. After 3 days the medium was changed and only EGF was maintained in the medium under all mode conditions. The medium was then changed twice weekly.

Isolation of human hematopoietic cells from HCO cultures

After at least 3 days of BMP pathway activation (e.g., day 9), HCO begins to contain vascular mesodermal cells expressing markers such as KDR, FLT1, and GATA 2. Continued growth in Matrigel resulted in the formation of hematopoietic endothelium expressing CD31, CD 34. Between day 15 and day 20, the cultures had endothelial tubes producing hematopoietic progenitor/stem cells expressing RUNX 1. The media from which the HCO cultures were collected identified a wide range of differentiated hematopoietic cells, including bone marrow cells (basophils, neutrophils, eosinophils) and monocytes and macrophages. Cells expressing immature B and T cell markers can also be observed using flow cytometry.

TABLE 21 day HCO Up-Regulation

Methocult^TMCell growth of the harvest medium in H4434 Classic resulted in the formation of colonies consisting of erythrocytes, granulocytes and macrophages. Methocult^TMH4434 Classic contains methylcellulose from Iscove's MDM, fetal bovine serum, bovine serum albumin, 2-mercaptoethanol, recombinant human Stem Cell Factor (SCF), recombinant human interleukin-3 (IL-3), recombinant human Erythropoietin (EPO), recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF).

Formation of functional macrophages in HCO

HCO was passaged into fresh Matrigel and culture continued, resulting in the formation of functional macrophages (from about day 20 to about day 34 or more). Macrophages are functionally responsive to infectious stimuli, such as Lipopolysaccharide (LPS) or bacteria, phagocytose the bacteria, and produce inflammatory cytokines, including IL6, IL8, CCL3, CCL4, and TNF- α, spontaneously and in response to LPS. Macrophages also respond to IL10, resulting in decreased inflammatory cytokine production. Macrophages also respond to M-CSF inhibition or addition with decreased and increased numbers of macrophages, respectively.

Method of generating human liver organoids with increased immune cell production

Differentiation of hipscs into definitive endoderm was induced using the previously described methods, with several modifications (Spence et al, 2011). Briefly, colonies of hipscs were isolated in Accutase (Thermo Fisher Scientific inc., Waltham, MA, USA) and 150,000-cell 300,000 cells were plated on Matrigel or laminin-coated tissue culture 24-well plates (Corning, Durham, NC). When the cells became high density (more than 90% of the wells were covered with cells), the medium was changed to RPMI 1640 medium (Life Technologies, Carlsbad, Calif.) containing 100ng/mL activin A (R)&D Systems, Minneapolis, MN) and 50ng/mL bone morphogenetic protein 4(BMP 4; r&D Systems) (on day 1), 100ng/mL activin a and 0.2% fetal calf serum (FCS; thermo Fisher Scientific Inc.) (at day 2), and 100ng/mL activin a and 2% FCS (at day 3). For days 4-6, cells were differentiated into the posterior midgut by culturing in Advanced DMEM/F12(Thermo Fisher Scientific Inc.), which contained 2% B27(Life Technologies), 1% N2(Gibco, Rockville, Md.), 2mM L-glutamine (Gibco) and 1mM HEPES (Gibco), 1% penicillin/streptomycin (Gibco), containing 500ng/ml fibroblast growth factor (FGF 4; R4; R.sup.&D Systems) and 3 μ M CHIR99021(Stemgent, Cambridge, MA, USA). Cultures for cell differentiation were maintained at 37 ℃ with 5% CO₂In a 95% air environment, the medium was changed daily. Differentiated definitive endoderm showed shoots on the plates at day 7. If insufficient spheroids were to be embedded in Matrigel, medium was added again on days 4-6 and incubated overnight at 37 ℃.

Differentiation into liver organoids the following four methods can be used to differentiate DE into liver organoids: "Matrigel drop method", "Matrigel sandwich method", "non-Matrigel method", and "sphere generation Transwell method", each of which is described below.

Matrigel drop method: on days 7-8, the definitive endoderm organoids with the coated cells were gently pipetted to layer from the dish. The separated spheroids were centrifuged at 800rpm for 3 minutes and, after removal of the supernatant, embedded in 100% Matrigel droplets on a petri dish. 250 μ L of Matrigel (Corning) was used per well of 24-well plates of endoderm cultures. An 80. mu.L Matrigel droplet was prepared, 1 droplet per well of a 24-well plate (VWR Scientific Products, West Chester, Pa.). Plates were incubated at 37 ℃ with 5% CO₂And placing the mixture in an environment of 95% air for 5-15 minutes. After the Matrigel solidified, 2. mu.M of Advanced DMEM/F12, and B27, N2, L-glutamine, HEPES, penicillin/streptomycin and retinoic acid (RA; Sigma, St. Louis, Mo.) were added and left for 1 to 5 days. The medium was changed every other day. After RA treatment, organoids embedded in Matrigel droplets were cultured in hepatocyte culture medium (HCM Lonza, Walkersville, Md.) with 10ng/mL hepatocyte growth factor (HGF; Peprotech, RockyHill, N.J.), 0.1. mu.M dexamethasone (Dex; Sigma) and 20ng/mL oncostatin M (OSM; R. RTM)&D Systems). Cultures for cell differentiation were maintained at 37 ℃ with 5% CO₂In a 95% air environment, the medium was changed every 3 days. The organoids embedded in the Matrigel droplets can be isolated by scraping and gentle pipetting for any analysis, about day 20-30.

Matrigel sandwich method: at days 7-8, the definitive endoderm organoids with the coated cells were gently pipetted to layer from the dish. The separated spheroids were centrifuged at 800rpm for 3 minutes, and after removing the supernatant, mixed with 100% Matrigel. At the same time, the hepatocyte culture medium with all supplements was mixed with the same volume of 100% Matrigel. The HCM and Matrigel mixture was applied to the bottom of the dish to form a thick coating (0.3-0.5cm) on the plate and at 37 ℃ in 5% CO₂And placing the mixture in an environment with 95% of air for 15-30 minutes. After the Matrigel was cured, spheroids mixed with Matrigel were seeded on a plate with a thick Matrigel coating. The plates were incubated at 37 ℃ in 5% CO₂In an environment of/95% airThe mixture was left to stand for 5 minutes. Add Advanced DMEM/F12, and 2. mu.M of B27, N2, L-glutamine, HEPES, penicillin/streptomycin and retinoic acid (RA; Sigma, St. Louis, Mo.) and left for 1-5 days. The medium was changed every other day. After RA treatment, organoids embedded in Matrigel droplets were cultured in hepatocyte culture medium (HCM Lonza, Walkersville, Md.) with 10ng/mL hepatocyte growth factor (HGF; Peprotech, RockyHill, N.J.), 0.1. mu.M dexamethasone (Dex; Sigma) and 20ng/mL oncostatin M (OSM; R. RTM)&D Systems). Cultures for cell differentiation were maintained at 37 ℃ with 5% CO₂In a 95% air environment, the medium was changed every 3 days. The organoids embedded in the Matrigel droplets were isolated by scraping and gentle pipetting for any analysis, about day 20-30.

Matrigel-free method: the definitive endoderm organoids with the coated cells were maintained for 4 days on days 7-8 in planar cultures with Advanced DMEM/F12(Thermo Fisher Scientific Inc.) and B27(Life Technologies), N2(Gibco, Rockville, Md.), L-glutamine, HEPES, penicillin/streptomycin and retinoic acid (RA; Sigma, St. Louis, Mo), (2 μ M). The medium was changed every other day. After 4 days of planar culture, organoids began to bud and 2D cells differentiated into hepatocytes. Both organoids and hepatocytes were maintained for more than 60 days in hepatocyte culture medium (HCM Lonza, Walkersville, MD) with 10ng/mL hepatocyte growth factor (HGF; Peprotech, RockyHill, NJ), 0.1. mu.M dexamethasone (Dex; Sigma) and 20ng/mL oncostatin M (OSM; R)&D Systems), 10 days. For organoid analysis, floating organoids can be collected in ultra-low attachment 6-well plates and used for subsequent analysis as appropriate. Cultures for cell differentiation were maintained at 37 ℃ with 5% CO₂In a 95% air environment, the medium was changed every 3 days.

The sphere generation Transwell method: a posterior midgut sphere was created as described above. The anterior foregut sphere was created by slight changes to d4-6 differentiation. For the anterior foregut spheres, Advanced DMEM/F12(Thermo Fisher Scientific Inc.) and B27(Life technologies), N2(Gibco, Rockville, Md.), L-glutamine, HEPES, penicillin/streptomycin, 500ng/ml FGF4, 2. mu.M CHIR99021 and 200ng/ml noggin were added and replaced daily for 4-7 days. On day 8, cells from the front and back were dissociated into single cell suspensions with pipetting and trypsinization and plated onto 96-well ultra-low attachment plates and cultured overnight. On day 9, cell aggregates were collected, front and back pooled and cultured overnight. On day 10, the front and back Matrigel drops attached to each other. 12-well plates (Denville) were coated with 50. mu.l Matrigel and incubated at 37 ℃ for 2 min. The attached spheroids of the anterior and posterior aggregates were carefully picked with a minimum amount of Matrigel using a large 10 μ L pipette and placed on a Matrigel coated 12-well plate. Another 5. mu.L of Matrigel was placed on each spheroid. Advanced DMEM/F12 and B27, N2, L-glutamine, HEPES and penicillin/streptomycin were added. On day 13, spheroids were collected with a large pore pipette and transferred to a transwell plate, covered with an additional 5 μ L Matrigel. Advanced DMEM/F12 was added to the bottom wells along with B27, N2, L-glutamine, HEPES and penicillin/streptomycin, and replaced every 5 days.

Embedded liver organoid cultures containing hematopoietic cells: at days 7-8, the definitive endoderm organoids with the coated cells were gently pipetted to layer from the dish. The cells were prepared by any of the methods described above as "Matrigel drop method", "Matrigel sandwich method" or "Transwell method for sphere generation". Cultures supplemented with Advanced DMEM/F12 and B27, N2, L-glutamine, HEPES and penicillin/streptomycin, without retinoic acid for cell differentiation were maintained at 37 ℃ and 5% CO₂The medium was changed every 4 days in a 95% air environment. Erythrocytes became visible in iPSC cultures on days 13-15. Thrombopoietin (TPO) (10ng/ml) and Stem Cell Factor (SCF) (100ng/ml) were added to the medium on day 7, increasing the production of hematopoietic cells (which can remain in the medium until collection).

Analysis of multiple hematopoietic lineages in vitro in liver organoid cultures: on days 8-18 of culture, organoids were dissociated by mechanical force by pipetting and washed with PBS. Cells were then treated with 0.05% trypsin-EDTA (Life technologies) to removeDemarigel and generate single cell suspensions. Cells were seeded on plates containing methylcellulose and cytokines including transferrin, Stem Cell Factor (SCF), interleukin 3(IL-3), interleukin 6(IL-6), Erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF) (stem cell technology) in a humidified chamber inside an incubator and maintained at 37 ℃ and 5% CO₂In a 95% air environment for 10 days, colonies including erythroid cells, macrophages and basophils were observed. Cells were identified by Wright-Giemsa staining.

Reference to the literature

Gomez Perdiguero, E.et al Tissue resident macrophages are derived from yolk sac-derived red-bone marrow progenitor cells (Tissue-responsive macrophages origin from yolk-sac-derived erythro-myogenic) Nature 518,547-551, doi 10.1038/Nature13989(2015).

Hoeffel, G, et al C-Myb (+) erythroid-myeloid progenitor cell-derived fetal monocytes produce adult tissue-resident macrophages (C-Myb (+) erythro-myoid progenisor-derived fetal monoclonal antibodies to induced tissue-responsive macrophages) immunization (Immunity) 42,665 678, doi 10.1016/j.immune.2015.03.011 (2015).

Sheng, J., Ruedl, C. & Karjalainen, K. Most Tissue Resident Macrophages, Except for Microglia, Are Derived from Fetal Hematopoietic Stem Cells (last Tissue-solvent macromolecules Exception Microglia Are Derived from foetal hematotic Stem Cells) < Immunity 43,382 393, doi:10.1016/j. Immunity 2015.07.016(2015).

De Schepper, S. et al, Self-sustained Intestinal Macrophages Are required for Intestinal Homeostasis (Self-maintenance Gut Macrophages Are area for Intestinal health) cells (Cell) 175,400-415e413, doi:10.1016/j. cell.2018.07.048(2018).

Development and maintenance of resident macrophages (The maintenance and maintenance of pathogenic macrogens) natural immunology (Nat Immunol) 17,2-8, doi:10.1038/ni.3341(2016).

Cumano, A., Dieterlen-Lievre, F. & Godin, I. Lymphoid potential probed before circulation in mice was limited to the embryonic wall within the tail embryo (Lymphoid potential, probe before circulation in motor, is restricted to the cardiac intracellular sporadic splanchnopleura) cells (1996) 86, 907-.

Bmp signaling is essential and sufficient for specification of Xenopus ventral endoderm development in Xenopus, Wills, a., Dickinson, k., Khokha, M. & Baker, j.c. Bmp signaling & ltb p signaling is a novel and supplementary for a novel endoderm specification) & ltdevelopment dynamics: a official publication (development dynamics: an of the American Association of Anatomists) 237, 2177-.

Tiso, n., Filippi, a., Pauls, s., Bortolussi, M. & Argenton, f.bmp signaling regulates the anteroposterior endoderm patterns in zebrafish developmental mechanisms (Mech Dev) 118,29-37(2002).

Roberts, D.J., et al, Sonic Hedgehog induced Endodermal signals of the Bmp-4and Hox Genes during Induction and division of the Hindgut in chickens Development 121,3163 3174(1995).

Munera, J.O. et al Human Pluripotent Stem Cells differentiate into colon Organoids (Differentiation of Human Pluripotent Stem Cells in vivo transduction of BMP signalling) Cell Stem Cells (Cell Stem Cells) 21,51-64e56, doi:10.1016/j.stem.2017.05.020(2017) by Transient Activation of BMP signals.

The role of BMP-4and GATA-2in The induced differentiation of hematopoietic mesoderm in Xenopus laevis (The role of BMP-4and GATA-2in The induction and differentiation of hematopoietic mesoderm in Xenopus laevis) Blood (Blood) 88,1965-1972(1996).

Iacovino, M. et al HoxA3 is a apical regulator of hematogenic endothelium (HoxA3 is an adaptive regulator of hematopoietic endipelium) Nature cell biology 13,72-78(2011).

Sturgeon, C.M., Ditadi, A., Awong, G., Kennedy, M. & Keller, G.Wnt signaling controls the specification of definitive primitive hematopoiesis from human pluripotent stem cells (Wnt signaling controls of the specificity of definitive and private hematology from human pluripotent cells) & Nature Biotechnology 32,554-561(2014).

Identification of hematopoietic endothelial progenitor cells and their direct precursors in human pluripotent stem Cell differentiation culture (Identification of the hematopoietic endogenous promoter and its direct precursors) Cell reports (Cell Rep) 2,553-567, doi:10.1016/j.cell 2012.08.002(2012).

The potential of Kennedy, M.et al human T lymphocytes marks the appearance of definitive hematopoietic progenitor cells in human pluripotent stem Cell differentiation cultures (T lymphocyte cellular potential marks of the experimental genes of definitive hematopoietic progenitors in human pluripotent stem Cell differentiation cultures) Cell report (Cell Rep) 2, 1722. sup. 1735, doi:10.1016/j.cell rep.2012.11.003(2012).

Bain, C.C. et al Resident and pro-inflammatory macrophages in the colon represent alternative environment-dependent fates of the same Ly6Chi monocyte precursors (origin and pro-inflammatory macrophages in the colon expression context dependent genes of the same Ly6Chi monocyte precursors) Mucosal (Mucosal Immunol) 6, 510, doi:10.1038/mi.2012.89(2013).

Continuous supplementation of circulating monocytes from Bain, C.C., et al maintained the macrophage pool in the intestine of adult mice (Constant repeat from circulating monocytes main aids in the gut of adult mice) Natural immunology (Nat Immunol) 15,929-937, doi:10.1038/ni.2967(2014).

Bain, C.C. et al Resident and pro-inflammatory macrophages in the colon represent alternative environment-dependent fates of the same Ly6Chi monocyte precursors (origin and pro-inflammatory macrophages in the colon expression context-dependent genes of the same Ly6Chi monocyte precursors) Mucosal immunology (Mucosal immunology) 6,498 510(2013).

Takata, K. et al, Induced Pluripotent Stem Cell-Derived Primitive Macrophages Provide a Platform for Tissue Resident Macrophage Differentiation and Function Modeling (Induced-pluratity-Stem-Cell-Derived conditional machinery provideda plan for Modeling Tissue-responsive mechanism Differentiation and Function) immunization (Immunity) 47,183-198.e186(2017).

Human proteomics for normal and cancer tissues based on antibody proteomics (a human protein atlas for normal and cancer tissues based on antibody proteomics) & molecular & cellular proteomics: MCP (Molecular & cellular proteins: MCP) 4,1920, 1932(2005).

Bulmer, J.N. & Johnson, P.M. Macrophage populations in the chorion of human placenta and amnion (Macrophage publications in the human placenta and amniochorion.) clinical and experimental immunology (Clin Exp Immunol) 57,393-403(1984).

Davies, L.C., Jenkins, S.J., Allen, J.E. & Taylor, P.R. Tissue-resident macrophages (Tissue-resident macrohages) Nature immunology (Nat Immunol) 14, 986. sup. 995, doi:10.1038/ni.2705(2013).

Tissue resident macrophages in the intestine of Shaw, T.N. et al have long lives and are determined by Tim-4and CD4expression (Tissue-responsive macrography in the interest area long and defined by Tim-4and CD4expression) journal of Experimental medicine (J Exp Med) 215, 1507-.

Human small intestine macrophage subsets were determined by Bujko, A. et al transcription and function profiling (trade and functional definitions human small intestinal macrophage subsets) J.Experil.Med.215, 441-458(2018).

Smith, P.D., et al, Intestinal macrophages lack CD14 and CD89 and therefore down-regulate LPS-and IgA-mediated activity (intracellular macromacrophages lack CD14 and CD89 and associated down-regulated for LPS-and IgA-mediated activities.) J.Immunol 167,2651-2656(2001).

TGF-beta2 inhibits macrophage cytokine production and mucosal inflammatory responses in the developing intestine (TGF-beta2 related to macrophage cytokine production and mucosal inflammatory responses) Gastroenterology (Gastroenterology) 140,242 and 253(2011).

Man, A.L. et al CX3CR1+ Cell Mediated Salmonella rejection Protects the Intestinal Mucosa at the beginning of Infection (CX3CR1+ Cell-Mediated Salmonella Exclusion Protects the Intestinal Mucosa for intracellular Mucosa Infection. J.Immunol.198, 335. sup. -343, doi: 10.4049/jimmmunol.1502559 (2017).

Martin, M.J., Muotri, A., Gage, F. & Varki, A. Human embryonic stem cells express immunogenic non-Human sialic acid (Human embryonic stem cells expressed in immunogenic non-Human sialic acid) & Nature & medicine (Nat Med) & 11,228- & 232, doi:10.1038/nm1181(2005).

Lancet, P.M., Gage, F.H. & Varki, A.P. Stem cell glycans (The polysaccharides of stem cells) New in chemical and biological (Curr Opin Chem Biol) 11,373-380, doi:10.1016/j.cbpa.2007.05.032(2007).

Glocker, e. -o. et al Inflammatory bowel disease and mutations affecting the interleukin-10receptor (inflammation and mutations affecting the interleukin-10 receptor): new england journal of medicine (N Engl J Med) 361,2033-2045(2009).

Tugimov, S.M. et al Differential transmission of HIV through fetal oral/intestinal epithelium and adult oral epithelium (Differential transmission of HIV transforming biological/endogenous and adolt oral epithelial) & J virology (J Virol) 86, 2556-.

Neutrophils migrating through Campbell, E.L. et al form a mucosal microenvironment through local oxygen depletion to affect resolution of inflammation (Transmigrating neutrophiles flap through calcium localized oxidation of inflammation) Immunity (Immunity) 40,66-77, doi: 10.1016/j.immunity.2013.11.020 (2014).

All percentages and ratios are by weight unless otherwise indicated.

All percentages and ratios are calculated based on the total composition unless otherwise indicated.

It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Rather, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "20 mm" is intended to mean "about 20 mm".

Each document cited herein, including any cross-referenced or related patent or application, is hereby incorporated by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it teaches, suggests or discloses any such invention alone or in any combination with any other reference. In addition, to the extent that any meaning or definition of a term herein conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to the term herein shall govern.

While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims (40)

1. A method of preparing a Hematopoietic Stem Cell (HSC) or cell derived therefrom, comprising

a. Contacting definitive endoderm derived from precursor cells with a wnt signaling pathway activator and an FGF signaling pathway activator until foregut cells are formed;

b. culturing said foregut cells in the absence of retinoic acid to form a liver organoid that produces hematopoietic cells.

2. The method of claim 1, wherein the precursor cells are selected from one or both of embryonic stem cells and induced pluripotent stem cells (ipscs).

3. The method according to claim 1 or 2, wherein the Wnt signaling pathway activator is selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, small molecule activators of the Wnt signaling pathway (e.g. lithium chloride; 2-amino-4, 6-disubstituted pyrimidine (hetero) arylpyrimidine; IQ 1; QS 11; NSC 668036; DCA β -catenin; 2-amino-4- [3,4- (methylenedioxy) -benzyl-amino ] -6- (3-methoxyphenyl) pyrimidine), WAY-316606; SB-216763; or BIO (6-bromoindirubin-3' -oxime)), siRNA and/or shRNA activators of the Wnt signaling pathway, GSK3 inhibitors (e.g., Chiron/CHIR9902), and combinations thereof.

4. The method of any one of the preceding claims, wherein the FGF signaling pathway activator is selected from the group consisting of small molecule or protein FGF signaling pathway activators, siRNA and/or shRNA activators of FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, FGF signaling pathways, and combinations thereof.

5. The method of any one of the preceding claims, wherein the foregut cells form spheroids prior to forming the liver organoid.

6. The method of any one of the preceding claims, wherein the foregut cells form spheroids prior to forming the liver organoid, and wherein the method further comprises disrupting the spheroids to form a plurality of cells.

7. The method of claim 6, wherein the disruption is by one or both of chemical disruption and mechanical disruption.

8. The method according to claim 6 or 7, wherein the disruption comprises treatment with an enzyme, preferably wherein the enzyme is an enzyme having one or both of proteolytic and collagenolytic activity, preferably one or more enzymes selected from accutase, trypsin, collagenase, hyaluronidase, deoxyribonuclease, papain, trypzen (manufactured by Sigma), or a combination thereof.

9. The method of any one of the preceding claims, wherein the foregut is cultured in the presence of a cytokine selected from the group consisting of transferrin, Stem Cell Factor (SCF), interleukin 3(IL-3), interleukin 6(IL-6), Erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), and combinations thereof, preferably wherein the foregut is dissociated into single cells prior to said culturing, wherein said culturing is performed with the cytokine for about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days, or about 21 days, or about three weeks, or about four weeks, or about five weeks, or about six weeks, or about seven weeks, or about eight weeks, or about nine weeks, or about 10 weeks, or about 11 weeks, or about 12 weeks, or greater than 12 weeks.

10. The method of any one of the preceding claims, further comprising contacting the human liver organoid with one or both of Thrombopoietin (TPO) and Stem Cell Factor (SCF), wherein said contacting with one or both of Thrombopoietin (TPO) and Stem Cell Factor (SCF) is for about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days, or about 21 days, or about three weeks, or about four weeks, or about five weeks, or about six weeks, or about seven weeks, or about eight weeks, or about nine weeks, or about 10 weeks, or about 11 weeks, or about 12 weeks, or greater than 12 weeks.

11. The method of any one of the preceding claims, wherein the human liver organoid is in a fetal state.

12. The method of any one of the preceding claims, wherein the human liver organoid produces reduced albumin compared to a human liver organoid treated with retinoic acid.

13. The method of any one of the preceding claims, wherein the human liver organoid produces alpha-fetoprotein (AFP).

14. The method according to any one of the preceding claims, wherein the human liver organoid has increased endothelial markers CD34 and KDR compared to a human liver organoid treated with retinoic acid.

15. The method of any one of the preceding claims, wherein the human liver organoid has increased Erythropoietin (EPO) and hemoglobin gamma (HBG) compared to a human liver organoid treated with retinoic acid.

16. The method of any one of the preceding claims, wherein the foregut cells are suspended in a basement membrane matrix (Matrigel).

17. The method of any one of the preceding claims, wherein the foregut cells are cultured on a stromal cell line from bone marrow.

18. The method of claim 1, wherein the derivative cells are selected from the group consisting of bone marrow cells (such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes and platelet-producing megakaryocytes), lymphoid cells (such as T cells, B cells and natural killer cells) and combinations thereof.

19. A method comprising

Culturing a colon organoid to form an organoid culture;

harvesting one or more immune cells from the colon organoid culture.

20. The method of claim 19, wherein the colon organoid is cultured for about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days, or about 21 days, or about three weeks, or about four weeks, or about five weeks, or about six weeks, or about seven weeks, or about eight weeks, or about nine weeks, or about 10 weeks, or about 11 weeks, or about 12 weeks, or more than 12 weeks, or until the colon organoid comprises one or more of hematopoietic endothelium and endothelial tubes that produce hematopoietic progenitor/stem cells.

21. The method of claim 19 or 20, further comprising isolating mesenchyme from the colon organoid culture and culturing the mesenchyme, preferably wherein the mesenchymal culturing step lasts for a period of about four days to three months, or about five days to two months, or about 6 days to about one month, or about seven days to about 21 days, more preferably wherein the mesenchymal culture is a suspension culture.

22. The method of any one of claims 19 to 21, wherein the colon organoid comprises mesenchyme, and wherein the culturing step lasts for a period of about four days to three months, or about five days to two months, or about 6 days to about one month, or about seven days to about 14 days, optionally wherein the culturing step is in suspension culture for a period of about one week to four weeks, or about one week to allow for extension of mesenchyme.

23. The method of claims 19-22, wherein the immune cells are selected from the group consisting of erythroid, myeloid, and mixed myeloid colonies.

24. The method of claims 19-23, wherein the immune cell is selected from one or more of a macrophage, a neutrophil, an eosinophil, a basophil, a red blood cell, a leukocyte, and a monocyte.

25. The method according to claims 19 to 24, wherein the colon organoid is derived from definitive endoderm derived from precursor cells, preferably embryonic stem cells or induced pluripotent stem cells.

26. The method of any one of claims 19 to 25, further comprising culturing with a T cell inducible growth factor.

27. The method of any one of claims 19 to 26, further comprising disrupting the culture to disperse the colon organoids and disrupting mesenchyme in the culture followed by culturing in basement membrane matrix for a period of about one week to about four weeks, or about two weeks to about three weeks.

28. A method of modelling a disease state selected from necrotizing enterocolitis, very early-onset IBD30, infection from bacterial pathogens (such as clostridium difficile), infection from viral pathogens (such as HIV, which is prone to infect fetal intestinal macrophages), the method comprising causing the disease state in a colon organoid prepared according to the method of any one of claims 19 to 27.

29. A method for modeling innate immunity mechanisms comprising a colon organoid prepared according to the method of any one of claims 19-27.

30. A Human Liver Organoid (HLO), characterized in that said HLO comprises fetal liver tissue, wherein said HLO produces hematopoietic cells.

31. A Human Colon Organoid (HCO) comprising a hematogenic endothelium.

32. HCO according to claim 30 or 31, wherein the hematogenic endothelium produces immune cells, preferably one or more of erythrocyte-myeloid progenitor cells, lymphoid progenitor cells and macrophages.

33. The HCO according to claim 31 or 32, wherein the hematogenic endothelium produces macrophages that secrete pro-inflammatory cytokines.

34. The HCO of claim 32, wherein the immune cell has bone marrow potential.

35. The HCO of claim 30, wherein the HCO comprises a hematopoietic progenitor cell having T cell potential.

36. The HCO of claim 30, wherein the HCO comprises hematopoietic progenitor cells, wherein the progenitor cells are CD34+, wherein the CD34 progenitor cells are in organoid mesenchyme, wherein the hematopoietic progenitor cells have the capacity to form T cells.

37. The method of any one of claims 30-36, wherein the HCO comprises endothelial tubes, wherein the ET is positive for CD34+, and wherein the ET comprises RUNX1+ cells.

38. A method of making HCO/HIO capable of producing Hematopoietic Stem Cells (HSCs), comprising contacting definitive endoderm derived from precursor cells with one or more factors for a sufficient period of time to produce mid/posterior intestinal spheroids, and optionally embedding the mid/posterior intestinal spheroids in a basement membrane matrix, and contacting the DE with a combination of factors comprising FGF, CHIR, noggin and SMAD inhibitor, in an amount and for a sufficient period of time to produce anterior foregut spheroids;

wherein the mid/posterior intestinal spheroids or anterior foregut spheroids produce HSCs.

39. A method of treating an individual in need of immune cells comprising

a. Harvesting Hematopoietic Stem Cells (HSCs) or derived cells from HCO or HLO according to any one of the preceding claims; and

b. administering the HSCs or their derived cells to an individual in need thereof, wherein the administering comprises transplanting the HSCs into the bone marrow of the individual.

40. The method of claim 39, wherein the treatment is of anemia (including aplastic anemia, Fanconi anemia), immunodeficiency, cancer (such as lymphoma, leukemia, carcinoma, solid tumors), hereditary diseases of hematopoietic function, hereditary storage diseases, thalassemia major, sickle cell disease, osteoporosis, or a combination thereof.

Images